Effect of cupric ions on the initiation protein synthesis rate in the human endometrium

CONTRACEPTION

EFFECT

OF CUPRIC IONS ON THE INITIATION PROTEIN RATE IN THE HUMAN ENDOMETRIUM

Omar Hernandez-Perez',

Griselda

SYNTHESIS

Lunal, Efrain Mercadol,

Nestor Delgado' and Adolf0 Rosado 3 1Laboratorio de Biologia Molecular, Unidad de Investigation Biomedica, Instituto Mexican0 de1 Seguro Social, Apartado Postal 73-032, Mexico, D.F. 03020, Mexico 2Unidad de Investigation Biomedica de Occidente, Mexican0 de1 Seguro Social, Guadalajara Jal Autonoma 3Universidad Mexico, D.F.

Metropolitana,

Unidad

Instituto Iztapalapa,

Abstract The effect of cupric ions on the initiation protein human endometrium was studied. rate of the synthesis complete ribosomal system copper to t e Addition of $ decreased the binding of [ HIMet-tRNA(i) to the isolated at about 70% inhibition with with a plateau ribosomes concentrations higher than 150 PM. initiation activity was GTP-dependent with a The This activity was very rapid, requiring 5 maximum at 2 mM. min to complete the reaction. Incubation of isolated factors with copper (300 PM) inhibited the initiation formation of the ternary complex. When the complete system was reconstituted with salt-washed ribosomes after ternary complex formation, no significant change on the inhibition observed. Addition of initiation factors to pattern was 5-min preincubated salt-washed ribosomes with 300 PM copper, after the elimination of excess copper, induced only a 12% on Met-tRNA(i) binding. This effect was not decrease modified by the presence of Sparsomycin, an elongation It was concluded that copper interferes with the inhibitor. the initiation process, probably at ternary complex formation level.

Submitted for publication May 31, 1989 Accepted for publication August 21, 1989

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Introduction It is known that the presence of copper enhances the contraceptive effect of an intrauterine device (IUD) (1). In clinical trials it has been clearly shown that copper not only increased contraceptive efficacy, but also decreased the expulsion rate and the occurrence of complications, such as hemorrhage (1). copper has been shown to inhibit the Intrauterine synthesis of DNA in the rat endometrium (2). It has also been shown that copper induces a significant decrease in the concentration of endometrial RNA (3) and a decrease in the The process of protein synthesis by this tissue (4) inhibition of RNA and protein synthesis is associated with a rapid loss of polysomes and accumulation of monosomes (3,5), defective protein chain initiation suggesting a (6). Further evidence about the possible role of copper ions as inhibitors of protein initiation can be found in some in vitro studies showing that preincubation in the presence of increasing concentrations of copper induced a progressive shift of the polysome pattern of the human endometrium toward the predominance of the lighter components with an increase in the concentration of ribosomal subunits (7). seems translational control of protein It that cells operates mainly at the synthesis in eucaryotic initiation step (6). Binding of Met-tRNA(i) to native 40 S units is the first step ribosomal in the process of initiation of protein synthesis in eucaryotic cells (8). This binding is mediated by a group of soluble proteic components called initiation factors. Different initiation factors which could bind Met-tRNA(i) have been described in the ribosomal KC1 wash, as well as in the cytoplasm of various eucaryotic cell types (9). One of these was called in a reaction eIF-2, which exclusively binds Met-tRNA(i) dependent on the presence of GTP (10). Formation of eIFcan be Z/GTP/Met-tRNA(i) ternary complexes studied by retention on Millipore nitrocellulose papers even in the absence of ribosomal particles (11). In order to further understand the mechanism of action of the copper-releasing IUD, we decided to assess the initiation capacity of the human endometrium under in vitro conditions closely related to those prevailing in the in vivo system. For this, we studied the effect of copper ions on the formation of the ternary complex between Met-tRNA(i), initiation factor eIF-2 and GTP, and on the transference of this complex to isolated endometrial 40 S subunits. Material

and Methods

were Endometrial biopsies described (5) from a group of

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obtained as 36 voluntary,

previously apparently

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healthy, regularly menstruating women between 25-35 years of taken during the secretory phase (20 + Biopsies were age. The endometrial tissue was 2 days) of the menstrual cycle. carefully rid of contaminating blood by washing the finely The tissue dispersed tissue with the homogenizing medium. was then carefully pat dried and weighed to the nearest mg The endonetrial tissue in a Roller-Smith torsion balance. was homogenized in IO volumes of ice-cold buffer A (20 mM KCl, 15 mM MgC12, 2 mM dithiothreitol, 50 mM henin and 20 mM HEPES-KOH, pH 7.4). from the postnitochondrial Ribosones were prepared supernatant containing 50 PM henin by the method of Lenz and Baglioni (12), diluting 1 ml of endonetrial homogenate with This is then overlayed on 4 ml 1 ml of ice-cold buffer A. of ice-cold 15% sucrose (w/w) in buffer A and centrifuged at 50,000 rpn for 3 hr in a Beckman 50 Ti angle rotor at 4OC. and the pellet is rinsed The supernatant is discarded, with ice-cold buffer A. Ribosones are stored at carefully They are diluted 3- to 4-6OOC in aliquots of 20-30 ~1. fold with buffer A just prior to use. Ribosones obtained as described above but suspended in 0.2 ml of buffer B (1 mM dithiothreitol, 6mM MgC12, 0.1 mM EDTA, 10% glycerol and 20 mM HEPES-KOH, pH 7.4) were used to initiation factors and salt-washed obtain unfractionated ribosones by the method described by Crystal et al. (13). Binding of (3H)Met-tRNA(i) (New England Nuclear) to isolated ribosones, unfractionated initiation factors (saltwash) and to initiation factors plus salt-washed ribosones was measured by the methods suggested by Lenz and Baglioni (12) in the absence and in the presence of copper ions. To study Met-tRNA(i) binding to complete ribosomes, from 30 to 300 pg of ribosomes, considering that 1 mg ribosome equals 14.0 optical density units at 260 nm, were incubated in 50 ~1 of buffer C (135 mM KOAc, 2 mM MgC12, 20 mM HEPES-KOH, pH 7.4) containing 0.1 FC (3H)Met-tRNA(i) (200 Mg(OAc)2, 1 mM pCi/pnole), 2mM GTP, 1 mM unlabeled nethionine; and one of the following CuC12 concentrations: 0, 15, 30, 75, 150 or 300 PM. Incubations, 7 min at 30°C, were initiated by the addition of tritiated Met-tRNA and stopped by the addition of 3 ml ice-cold buffer C. After nixing, the content of the tubes was filtered through nitrocellulose filters, presoaked in ice-cold buffer C, as described by Nirenberg and Leder (11). Filters are then washed three times with 3 ml of ice-cold buffer C. The filters were then dried under a heat lamp and counted with 5 ml aquasol in a Packard Tricarb, Model 3390, liquid scintillation spectrometer with an efficiency of 60%, as determined by the channel ratio method.

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activity binding of the initiation The Met-tRNA(i) factors can be directly measured by a nitrocellulose filter binding technique similar to that described above. For this, 50 ~1 of salt-wash containing 225 pg of unfractionated initiation factors were incubated in 0.1 ml of buffer HEPESKOH, pH 7.4, containing from 0 to 3 mM GTP, 1 mM Mg(OAc)2 methionine. mM unlabelled The mixture was and 1.0 preincubated with different concentrations of CuC12 during 5 min at 30°C (time established in pilot experiments) and then 0.1 )LC (3H)Met-tRNA(i) was added to initiate the reaction. Incubations were made for 7 min at 30°C. After this time, the reactions were stopped by the addition of 1.5 ml ice(30 mM HCl, 20 mM Mg(OAc)2, 2 mM cold buffer D dithiothreitol, 20 mM HEPES-KOH, pH 7.4), and the bound MettRNA(i) was determined by filtration through nitrocellulose filters as described above. To discriminate if copper acts on the initiation factors and/or on the ribosomal subunits, some experiments were run with salt-washed ribosomes preincubated for 5 min at 30°C with several concentrations of CuC12 and then washed once by centrifugation, to remove unbound ions, copper before the addition of the initiation factors. To demonstrate that copper acts only on the initiation step of protein synthesis, some experiments were run in the as inhibitor of protein presence of 0.1 mM Sparsomycin elongation (14). Sparsomycin was a gift from the National Institutes of All other reactants were obtained Health. from Sigma Chemical Co. (St. Louis, Missouri). Results The ability of the ribosomal KC1 wash to bind MettRNA(i) in the presence of GTP was taken as a criterion that it contained eIF-2 activity (Fig. 1C). With the presence of Met-tRNA(i) GTP, binding of optimal concentration of The reaction was completed proceeded very quickly at 30°C. after 5 min (Fig. 1B). GTP was required, but concentrations higher than 2 mM were inhibitory (Fig. 1A). The rate of protein synthesis initiation in the human endometrium was significantly decreased by the presence of Addition of copper ions to tge complete copper (Fig. 2). ribosomal system induced a rapid decrease on ( H)Met-tRNA This decrease was incorporation to the isolated ribosomes. reaching a linear with small concentrations of copper, plateau at about 70% inhibition when copper concentrations were higher than 150 PM; 15 pm copper induced 25% inhibition The effect of copper on of initiation activity (Fig. 2). initiation was not modified by the presence of Sparsomycin (Fig. 3).

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8_

A

I

I

l!O do

I

3!omM

(GT PI

Time

(mid

-+-+-++NO

GTP

Fig. 1. Some characteristics of Met-tFWA(i) binding to the ribosomes. Fig. 1A shows effect of salt-washed the different concentrations of GTP with a maximum of 2 mM. Fig. 1B shows the time course of the reaction, indicating a very rapid process at 30°C with a time to complete the reaction of 5 min. Fig. 1C indicates the dependence of Metbinding on concentration (75-375 wg) of tRNA(i) the unfractionated initiation factors. In all cases, each point represents the mean + SD of at least 5 experiments.

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-I-

30

1 75

150

360JlNl

(CuCl2)

Inhibitory effect of several copper concentrations Fig. 2. on the binding of Met-tRNA(i) to the complete ribosomal This effect was linear at low concentrations of system. copper, reaching a plateau at about 70% inhibition at copper concentrations higher than 150 WM. Each point represents the mean k SD of at least 3 experiments.

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f

T

-ml A

6

6

B

C

6

D

Inhibition of ternary complex formation by cupric Fig. 3. B: Met-tRNA(i) binding to unfractionated ions. A: Control. initiation factors in the presence of 300 FM copper. C: with salt-washed ribosomes Complete system reconstituted after ternary complex formation in the presence of copper. D: Addition of initiation factors to 5-min preincubated, ribosomes with 300 GM salt-washed after the copper, elimination of excess copper. E: Complete system, as D, in the presence of 0.1 mM Sparsomycin. Bars indicate the mean + SD with the number of cases indicated inside the bars. Differences of all other bars (B, C, D and E) with respect to the control value (bar A) were statistically significant (p ~0.05) using t test for unpaired samples. Differences between D and E bars and between B and C bars were not significant.

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Incubation of isolated initiation factors in the presence of copper induced a rapid decrease on ( H)MettRNA(i) incorporation into the nitrocellulose paper-retained radioactivity and, therefore, on the formation of the ternary complex (Fig. 3B). When the complete system was reconstituted with salt-washed ribosomes after allowing the formation of the ternary complex in the presence of copper ions, no significant change on the inhibition pattern was observed (Fig. 3C). If the excess copper is eliminated by centrifugation previous to the addition of the soluble fraction, preincubation for 5 min of salt-washed ribosomes in the presence of copper concentrations as high as 300 )IM induced only a small, although significant, decrease on Met-tRNA(i) binding (Fig. 3D). This effect was not modified when elongation of the protein chain was inhibited by the presence of Sparsomycin in the incubation system (Fig. 3E). Discussion As is well known, the initiation mechanism of protein synthesis implies a series of reactions involving ribosomal subunits, protein factors, initiator tRNA, mRNA and GTP The first step consists of the binding of initiator (15). factor eIF-3 to the 40 S ribosomal subunits preventing its reassociation with the 60 S ribosomal subunit until MettRNA(i) binds to the 40 S subunit on the donor or peptidyl site. Binding of Met-tRNA(i), the second step, requires the formation of a stable ternary complex between eIF-2, GTP and Met-tRNA(i) in that order of sequence and concludes with the binding of this complex to the 40 S ribosomal subunit in an interaction that does not require the AUG initiation codon or mRNA. In the third step, mRNA binds to the 40 S preinitiation complex, requiring the presence of factors eIF-4A and eIF-4B as well as ATP. Finally, the 60 S subunit joins the 40 S complex to form the SO S ribosomal unit. This interaction requires the activation of the ribosomedependent GTPase whose activity mediates the dissociation of factor eIF-2 and eIF-3 prior to coalescence of the 60 S and 40 S subunits. Our results show that concentrations of copper as low as 15 PM--and therefore within the range of copper concentrations found in the endometrium of women using a copper-releasing IUD (16,17)--are capable of inducing a significant decrease on the formation of the initiation complex of protein synthesis (Fig. 2). These results are in agreement with previous observations showing copper ions inhibit protein synthesis in the human endometrial tissue (2,4), inducing an increase in the concentration of unbound mRNA and tRNA, a decrease in RNA and RNP-particles (5); and also with the in vitro studies showing that addition of

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increasing concentrations of copper induced a progressive components of rabbit polysome the heavy decrease of endometrial homogenates, with a concomitant increase in the lighter components and in the amount of ribosomal subunits (7) * Comparison of the results obtained by the addition of copper to the whole ribosomal system with those obtained by incubation of only the isolated initiation factors (Fig. 2) supports the idea that copper interferes mainly with the soluble part of the system, inhibiting the formation of the ternary complex Met-tFWA(i)/eIF-2/GTP, and that the effect ribosomal subunits, although metal over the of this significant, is less important. Binding of the ternary complex to 40 S subunits and finally to the large ribosomal subunit depends, at least in part, on the production of conformational changes in the 40 S subunit (6). The facility with which this conformational change occurs depends on the presence of free sulfhydryl groups on this subunit (18). Blockage of these functional copper will explain the small, although groups by inhibition produced by the pre-incubation of significant, These results salt-washed ribosomes with copper (Fig. 3). may also be explained by the finding that the presence of induces a significant decrease in the magnesium copper concentration of the microsomal fraction (19). This decrease would make the formation of functioning 80 S units difficult. The functional active unit in protein synthesis is the polysome, and its normal functioning in the endometrium is the regulated by alternate actions of estrogens and progestogens which integrate a cycle involving mutual effects over many indispensable processes that require protein synthesis: the synthesis of hormonal receptors (20), the induction of enzyme activities (21,22), preparations for implantation through the synthesis of specific proteins (23), etc. It would appear that the presence of copper in the endometrial environment interferes with this functional cycle, impairing the normal protein synthetic process of this tissue and, therefore, its physiological activity. References 1.

Kivijarvi A, Timonen H, Hirvonen E, Kajanoja copper IUDs: one year multicenter trial. Deliv Syst Monograph II 1985:302-g.

2.

Hagenfeldt K. Studies on the mode of action of the copper-T device. Acta Endocrinol 1972 (Suppl); 169:924.

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Hicks JJ, Hernandez-Perez 0, Aznar R, Mendez JD, Rosado A. Effect of intrauterine copper on the nucleic acids, polysome pattern, and glycoprotein composition of the human endometrium. Am J Obstet Gynecol 1975;121:981-6.

4.

Prager R. Effect of various types of,~ntss;te;~;~ incorporation foreign bodies on the mucoprotein and of thymidine -2-14C into DNA of rat's Fertil Steril 1972;20:944-50. endometrium.

5.

Hernandez-Perez 0, Aznar R, Ballesteros LM, Mendez JD, Rosado A. RNA metabolism of the normal and the coppertreated human endometrium. Contraception 1976;14:42133.

6.

Lodish H. Translational control Annu Rev Biochem 1976;45:39-45.

7.

Hernandez-Perez 0, Ballesteros LM, Mendez JD, Rosado A. Copper as a dissociating agent of liver and endometrial polysomes. Fertil Steril 1974;25:108-12.

8.

Darnbrough CH, Legon S, Hunt T, Jackson RJ. Initiation of protein synthesis: evidence for messenger RNAindependent binding of methionyl-transfer RNA to the 40s ribosomal subunit. J Mol Biol 1973;76:379-403.

9.

Mazumder R, Szer W. Protein biosynthesis. In: Florkin A, VanDeenen LLM, eds. Comprehensive M, Neuberger biochemistry. Vol. 24. Amsterdam: Elsevier, 1977;186233.

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Miller DL, Weissbach H. Factors involved in the transfer of aminoacyl-tRNA to the ribosome. In: Weissbach H, Petska S, eds. Molecular mechanisms of protein synthesis. New York: Academic Press, 1977;32373.

11.

Niremberg MW, Leder P. RNA codewords synthesis. Science 1964;145:1399-1407.

12.

Lenz JR, Baglioni C. regulation of Met-tRNA 1979;60:281-9.

13.

Crystal RG, Elson NA, Anderson WF. globin synthesis: assays. Meth Enzymol

14.

Herner AE, Goldberg IH, Cohen LB. Stabilization of Nacetylphenylalanyl tRNA binding to ribosomes by Sparsomycin. Biochemistry 1969;8:1335-43.

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Assays for investigating the binding activity. Meth Enzymol Initiation of 1974;30:101-12.

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Initiation of messenger RNA translation into Revel M. protein and some aspects of its regulation. In: of Weissbach H, Petska S, eds. Molecular mechanisms York: New Academic Press, protein biosynthesis. 1977;246-321

16.

Molecular distribution of trace Hicks JJ, Rosado A. metals in the normal and in the copper-treated human Internat J Fertil 1976;21:55secretory endometrium. 60.

17.

Hefnawi F, Kandil 0, Aksalani H, Zaki K, Nasr F, Mousa M. Copper levels in women using intrauterine devices Fertil Steril 1974;25:556-61. or oral contraceptives.

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Steinert PM, Baliga BS, Munro HN. Available groups of mammalian ribosomes in different states. J Mol Biol 1974;88:895-911.

19.

Hernandez-Perez 0, Aznar R, Hicks JJ, Ballesteros LM, Rosado A. Subcellular distribution of trace metals in the normal and in the copper-treated human secretory endometrium. Contraception 1975;11:451-64.

20.

Katzenellenbogen receptor action.

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Rosado A, Mercado E, Gallegos AJ, de 10s Angeles Wens R. Subcellular distribution of lysomal M, Aznar enzymes in the human endometrium. I. Normal menstrual cycle. Contraception 1977;16:287-298.

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Miyazaki K, Miyamoto E, Maeyama M, Uchida M. Specific regulation by steroid hormones of protein kinases in the endometrium. Eur J Biochem 1980;104:543-47.

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O'Grady JE, Bell SC. The blastocyst implantation. Development in mammals. Holland, 1977;185-245.

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BS. Dynamics of steroid hormone Annu Rev Physiol 1980;42:17-35.

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